Metabolic engineering of Cupriavidus necator for heterotrophic and autotrophic alka(e)ne production

Abstract

Alkanes of defined carbon chain lengths can serve as alternatives to petroleum-based fuels. Recently, microbial pathways of alkane biosynthesis have been identified and enabled the production of alkanes in non-native producing microorganisms using metabolic engineering strategies. The chemoautotrophic bacterium Cupriavidus necator has great potential for producing chemicals from CO2: it is known to have one of the highest growth rate among natural autotrophic bacteria and under nutrient imbalance it directs most of its carbon flux to the synthesis of the acetyl-CoA derived polymer, polyhydroxybutyrate (PHB), (up to 80% of intracellular content). Alkane synthesis pathway from Synechococcus elongatus (2 genes coding an acyl-ACP reductase and an aldehyde deformylating oxygenase) was heterologously expressed in a C. necator mutant strain deficient in the PHB synthesis pathway. Under heterotrophic condition on fructose we showed that under nitrogen limitation, in presence of an organic phase (decane), the strain produced up to 670mg/L total hydrocarbons containing 435mg/l of alkanes consisting of 286mg/l of pentadecane, 131mg/l of heptadecene, 18mg/l of heptadecane, and 236mg/l of hexadecanal. We report here the highest level of alka(e)nes production by an engineered C. necator to date. We also demonstrated the first reported alka(e)nes production by a non-native alkane producer from CO2 as the sole carbon source.

Review of NAD(P)H-dependent oxidoreductases: Properties, engineering and application

“…These enzymes are able to form long-chain aldehydes by reduction of fatty acids bound to acyl carrier protein (ACP) and/or to CoA, the latter case being similar to the CoAdependent aldehyde dehydrogenases described above, but acting on long-chain substrates. Some of the enzymes of this group prefer acylCoA as the substrate, while others act more efficiently on acyl-ACP substrates, so will generate products from CoA-dependent fatty acid beta-oxidation or ACP-dependent fatty acid synthesis pathways, respectively, both of which are being investigated through metabolic engineering [101,102]. Interestingly, some of them are able to reduce fatty acid acyl-CoA/ACP directly to long-chain alcohols, requiring oxidation of two molecules of NAD(P)H, instead of one [103].…”

Hydrogen‐Driven Cofactor Regeneration for Stereoselective Whole‐Cell C=C Bond Reduction in Cupriavidus necator

“…www.chemsuschem.org concentration of 67.7 gL À1 over ap eriod of 72 h. [13] Lonsdale et al reported as imilarr ecombinant system in which the same SH was coupled to aP 450 monooxygenase in Pseudomonas putida, [17] providing at hreefold increase in yield compared to the uncoupled system, with their reported yields rising from 36 to 101 mg L À1 .W hereas the heterologous co-expression of hydrogenases can be quite complex,t he direct use of C. necator offers the possibility of utilizing nativelye xpressed hydrogenases in their natural environment. [23] In summary,w eb elieve that the system proposed in this work lays important groundwork towards the utilization of the hydrogen-oxidizing C. necator as ar obust biocatalytic platform. Thisa pproach has been used by Grousseau et al forthe productiono f3.4 gL À1 isopropanol.…”

Alka(e)ne synthesis in Cupriavidus necator boosted by the expression of endogenous and heterologous ferredoxin–ferredoxin reductase systems

“…This second amplification was carried out using a forward primer (3) and a reverse primer (4) The ferredoxin‐NADP reductase gene (H16_B0102) was amplified from C. necator H16 genomic DNA using a forward primer (5) and a reverse primer (6) A synthetic ribosome binding site (RBS) sequence was encoded in the forward primers (1) and (5) and thus incorporated upstream of each gene. The RBS sequence (5′‐ AAAGGAGGACAACC ‐3′) was already used in a previous work (Crépin et al, ). The pLC10 plasmid (Crépin et al, ) used as a backbone was digested by Hin dIII and treated with Antarctic phosphatase.…”

“…Consequently, several pathways were reported for engineering alkane and alkene production derived from fatty‐acid metabolism. Recently, we successfully expressed an engineered alka(e)ne pathway in C. necator based on a cyanobacterial native pathway (Crépin et al, ). In cyanobacteria, two enzymes, an acyl‐ACP reductase (AAR, EC:1.2.1.80) and an aldehyde deformylating oxygenase (ADO, EC:4.1.99.5), constitute the alkane biosynthetic pathway (Figure ) (N. Li et al, ; Schirmer, Rude, Li, Popova, & del Cardayre, ; Warui, Li, Nørgaard, Krebs, & Booker, ).…”